In fiscal year 2018 we have conducted studies on the effects of interactions of inoculated retroviruses (exogenous retroviruses) regarding their replication after sequential inoculation of retroviruses in mice. In vitro cell cultures infected by a retrovirus are profoundly resistant to further infection due to blockade of cell surface receptors; a phenomenon termed retroviral interference. In vivo retroviral interference studies present some perplexing results. Protection of mice from retroviral-induced disease by prior infection of another retrovirus appears to occur by a receptor-blockade mechanism but is conferred early, before extensive infection by the blocking virus might be expected. This study quantified the extent of suppression of the challenge virus and the level of infection by the blocking virus at the time of challenge. Our results indicate that complete suppression of challenge virus replication can be achieved at minimal levels of infection by the blocking virus. These results suggest that widespread infection of the host requires the initial infection of a very small population of cells and that infection of this population by the blocking virus confers resistance to further infection by receptor blockade. Regarding the interactions of inoculated viruses with their endogenous counterparts we have previously shown in mice infected with the exogenous ecotropic Friend MuLV (F-MuLV) that endogenous polytropic transcripts not only recombine with F-MuLV but are mobilized by packaging of complete transcripts within F-MuLV virions in the absence of recombination. These pseudotyped endogenous polytropic transcripts, consisting of severely deleted as well as nearly complete genomes, are transferred by infection and integration into the genome of new cells. Normally the expression of endogenous retroviruses is under tight control by several processes, however upon infection of new cells we speculate that the control may be less stringent. We are currently examining this possibility. In FY 2018 we have continued studies on the interactions of retroviruses with host factors that restrict retroviral replication. Both mice and humans express APOBEC3 proteins (mA3 and hA3G, resp.) that have cytidine deaminase activity leading to G to A hypermutation of viral transcripts and inactivation of infecting retroviruses. We have found that mA3 packaged in virions results in a loss in the specific enzymatic activity of the viral polymerase and specific infectivity. This is accompanied by a substantial increase in the overall mutation rate, however, we did not observe G to A hypermutation. Thus, the inhibitory action of mA3 on most MuLVs is unlikely to involve cytidine deamination. It has been reported by one group that the ecotropic MuLV, AKV, in contrast to other MuLVs, undergoes G to A hypermutation when mA3 is packaged within AKV virions in human 293T cells. We have found that maximal levels of hypermutation of AKV occurs in mouse 3T3 cells in the absence of added mA3 and is the result of the expression of small amounts of endogenous mA3 in a portion of the cells. Furthermore, in contrast to AKV, the deaminase-independent action of mA3 on another MuLV was found to be dose-responsive indicating distinct mechanisms of action. NIH 3T3 cells are commonly employed for in vitro mutational studies. The finding that very low levels of endogenous mA3 in NIH 3T3 cells could mediate maximal levels of hypermutation of an MuLV was unexpected and illustrate the importance of considering the contribution of endogenous restriction factor expression in mutational studies employing in vitro cell lines. Previous work on the effect of endogenous MuLVs on autoimmune disease focused on murine lupus nephritis. In particular, on the characterization of the endogenous retroviral envelope glycoprotein, gp70, implicated in disease. This protein is secreted by hepatocytes as an acute phase protein and was believed to be a product of an endogenous xenotropic virus. Our studies revealed that endogenous polytropic viruses provide important sources of serum gp70 involved in lupus nephritis and their expression is under tight control by several genetic loci. We have recently initiated a collaborative investigation with Dr. Yang Dai of the Scripps Research Institute on the role of endogenous retroviruses in Type 1 diabetes. Type 1 diabetes in humans and in the Non Obese Diabetic (NOD) mouse strain is characterized by autoimmune destruction of the insulin-producing beta cells in the pancreas primarily by autoreactive T cells. The cause of Type 1 diabetes is unknown; however, it is believed to involve a combination of genetic and environmental factors. A major genetic contribution to development of Type 1 Diabetes in humans comes from a locus termed IDDM1, which consists of several genes located in the Major histocompatibility complex on chromosome 6. Within this region are several human endogenous K-type retroviruses (HERV-K) elements that segregate with genes that influence the development of the disease. Dr. Dai has found that Type 1 diabetes in the NOD strain is associated with the expression of endogenous retrovirus envelope (Env) and core proteins (Gag) in the mice. Our role in this collaboration is to identify the source of these proteins. Prelimary sequence analyses done by Dr. Dai, suggests that subtle variations in the Gag-encoding sequences may be the result of endogenous virus replication. Our initial experiments will be focused on the identification of replicating retroviruses and determining if endogenous viruses are mobilized in these mice.